The Extracellular Matrix: Composition and Structure
The extracellular matrix (ECM) is a complex composite of proteins, glycoproteins and proteoglycans (PGs). Awareness of this complexity has been heightened by the recognition that ECM components, individually or in concert with each other or other extracellular molecules, profoundly influence the biology of the cell and hence of the physiology of the whole structure in to which the cell is embedded. The functions of the ECM described so far are many but can be simply categorised as control of cell growth, providing structural support and physical stabilization, affecting cell differentiation, orchestrating development and tuning metabolic responses (42).
PGs are a family of heterogeneous and genetically unrelated molecules. The number of full-time as well as part-time members is constantly expanding. The terms ‘full-time’ and ‘part-time’ refer to the fact that some known PGs can exist as glycoproteins and some proteins can be found in a glycosylated form. In general, PGs are composed of a core protein to which one or more Glycosaminoglycan (GAG) chains are covalently attached by N or O linkage. GAGs are highly anionic linear heteropolysaccharides made of a disaccharide repeat sequences (53). However, there have been reports of PGs devoid of the GAG side chain (4; 106). GAGs can be classified into four distinct categories based on their chemical composition (53). The first category is the chondroitin/dermatan sulphate (CS/DS) chain consisting of alternating galactosamine and glucuronic/iduronic acid units. A second class, which is by far the most structurally diverse, is the heparin/heparan sulphate (H/HS) group which is composed of alternating glucosamine and glucuronic/iduronic repeats. The third type is the glucosamine and galactose containing keratan sulphate (KS) GAG. Hyaluronic acid (HA) is composed of glucosamine and glucuronic acid repeats. It is the most distinct GAG since it is not sulphated and is not covalently linked to the core protein of PG. Instead, HA binding to the PG core protein is mediated by a class of proteins known as HA binding proteins which exist in the ECM, on the cell surface and intracellularly (93).
Perlecan is a large HSPG with a core protein size of 400-450 kDa known to possess three HS chains. It was first isolated by Hassell et al. (44). It acquired its name from its appearance in rotary shadowing electron microscopy where it looks like a pearl on a string. It is a large multi-domain protein and thus one of the most complex gene products (23; 52).
Domain I is the N-terminus, this containing acidic amino acid residues which facilitate the polymerisation of heparan sulphate (52). However, recombinant domain I has been shown to accept either HS or CS chains; an observation that has been confirmed by in-vitro studies characterizing PGs synthesized in response to transforming growth factor β (TGF-β) and foetal calf serum showing that perlecan can be synthesized with CS chains (13). Ettner et al. (26) have shown that the ECM glycoprotein laminin, binds to perlecan domain I, as well as domain V both of which can carry the HS side chain. Loss of the HS chain abolished the binding.
Globular domain II was postulated to mediate ligand binding by the low-density lipoprotein (LDL) receptor due to their homology (30; 79). Heparitinase treatment abrogates this interaction pointing to the fact that the HS GAG side chains are involved in the binding (30).
Domain III of perlecan contains an RGD tripeptide sequence that provides a binding capacity for integrin receptors and provides anchorage for the 19, cell (18). Yamagata et al. have shown using double-immunofluorescence that perlecan colocalizes with integrins in cultured fibroblasts (104). This domain has also been shown to be homologous to the laminin short arm (51).
Domain IV is the largest domain of perlecan containing a series of immunoglobulin (Ig)-like repeats similar to those found in the Ig superfamily of adhesion molecules leading to the speculation that it may function in intermolecular interactions (47). Finally, domain V possessing three globular domains homologous to the long arm of laminin is thought to be responsible for self-assembly and laminin mediated cell adhesion (14).
The multiplicity and variety of perlecan's structural domains are indicative of its potential functions. Perlecan, in addition to binding to laminin and integrins, has been shown to bind fibronectin via its core protein (51). The HS chains of perlecan also have a very important functional role which has proven to be diverse. It has been reported that perlecan mediates the interaction between skeletal muscle cells and collagen IV via the HS GAG side chain (98). Recent studies have led to the identification and characterization of perlecan as a ligand for L-selectin in the kidney (65). Whether this interaction is via the core protein and/or the HS side chain is not clear. The group of Varki has identified in a series of experiments the HS GAG as well as heparin from endothelial cells as a ligand for both L- and P-selectins but not E-selectins (59; 80). The HS side chains in general, and those attached to perlecan core protein in particular, are known to bind growth factors such as fibroblast growth factors (FGF)-2, FGF-7, TGF-β, platelet factor-4 and platelet-derived growth factor-BB (PDGF-BB) (31; 52). The functional significance of these interactions has been highlighted by numerous studies demonstrating the role of perlecan in angiogenesis (5; 87), the control of smooth muscle cell growth (10) and the maturation and maintenance of basement membranes (19). The functional importance of perlecan has been demonstrated by a study of mice lacking perlecan gene expression (19). Homozygous null mice died between embryonic days 10 and 12. The basement membranes normally subjected to increased mechanical stresses such as the myocardium lost their integrity and as a result small clefts formed in the cardiac muscle leading to bleeding in the pericardial sac and cardiac arrest. The homozygotes also had severe cartilage defects characterised by chondrodysplasia despite that fact that it is a tissue which normally lacks basement membrane. This finding was interpreted as a potential proteolysis-protective function for perlecan in cartilage (19). The delay in detecting abnormalities until E10 suggests a certain redundancy with compensatory molecules being able to substitute for perlecan such as the basement membrane HSPGs collagen XVIII (38) and agrin (36).
Large aggregating PGs are, to date, composed of four members; versican, aggrecan, neurocan and brevican (52). The hallmark of these PGs is the ability to bind hyaluronic acid forming highly hydrated aggregates. They are also characterized by their tridomain structure composed of an N-terminal domain where HA binding occurs, a central domain carrying the GAG side chains and lectin binding C-terminus.
Versican is a PG with a core protein of 265-370 kDa which was originally isolated from human fibroblasts and is the homolog of the avian PG-M (110). It can possess 10-30 chains of CS and has been also reported to carry KS GAG chains (109). It is expressed by keratinocytes, smooth muscle cells of the vessels, brain and mesengial cells of the kidney. The N-terminal domain is responsible for the hyaluronic acid binding properties of versican (61). The central domain of versican consists of the GAG binding subdomains, GAG-α and GAG-β. These subdomains are encoded by two alternatively spliced exons and this gives rise to different versican isoforms. To date four isoforms have been recognized. V0 contains both GAG-α and GAG-β. V1 and V2 are known to possess domain GAG-β and GAG-α respectively (109). V3 is the variant which contains neither of the two subdomains and hence carries no CS/DS GAG side chains and has been localized in various mammalian tissues (63; 82; 105). The third domain of versican is the C-terminus and consists of a lectin-binding domain, an EGF-like domain and a complement regulatory protein-like domain. This C-terminus binds the ECM glycoprotein, tenascin (3), heparin and heparan sulphate (88) and fibulin (2). Versican is known to have an inhibitory effect on mesenchymal chondrogenesis (108), promotes proliferation (107) and migration via the formation of pericellular matrices via its interaction with cell surface bound hyaluronic acid (27). The formation of pericellular matrices is not only achieved via the core protein association with HA but also through GAG side chain interaction with the cytoskeletal associated cell surface receptor, CD44 (55). The postulated role of versican in migration has been also further reinforced by the recent findings of its interaction with both L- and P-selectins via the CS/DS side GAG chains (56). Furthermore, versican GAG side chains modulate chemokine response (45) and has been recently reported to possess growth factor binding capacity (111) and binding to β1 integrin Wu, Chen, et al. 2002 394.
Aggrecan is another large aggregating proteoglycan. It is known to be a major structural component of cartilage. It is composed of three globular domains and two GAG attachment domains (100). The N-terminal globular domain (G1) binds HA and link protein to form large aggregates. The second globular (G2) domain is unique to aggrecan and has no HA binding capacity. The function of this domain has not been clearly defined. The interglobular domain between the G1 and G2 contains proteolytic cleavage sites for metalloproteinases and thus been heavily investigated in pathologies where degradation of this domain is a hallmark, such as osteoarthritis. A KS domain is located at the C-terminus of the G2 domain followed by the CS domain. The CS domain is the largest domain of aggrecan and the domain which contributes to the hydrated gel-like forming capacity of aggrecan and thus its importance in load-bearing function. The last domain is the globular domain (G3) which contains three modules: an epidermal growth factor-like domain, a lectin module and a complement regulatory module. This domain is responsible for the interaction of aggrecan with the ECM glycoprotein, tenascin.
Functions of Extracellular Matrix Proteoglycans
In addition to contributing to the mechanical properties of connective tissues, extracellur matrix (ECM) PGs have biological functions which are achieved via specific classes of surface receptors. The two main classes are the syndecan and integrin receptor families (42). However, other receptors have also been described to bind ECM components such as the selecting family of glycoproteins (80), CD44 with all its variants (33), cell surface enzymes such as hyaluronic acid synthases (89), and PGs (52). The effects of the ECM do not and cannot, in an in vivo milieu, ever occur without the influence of other molecules. This statement is based on two well-described concepts. The first being that part of the effects of growth factors, cytokines, hormones and vitamins, as well as cell-to-cell contact and physical forces is alteration of the ECM production. The second concept is that the effects of the ECM on the cell bear a striking similarity to those effects observed in response to the above mentioned factors. This is a phenomenon known as “mutual reciprocity” (42) which is an oversimplified view of a complex set of modular interactions, i.e. as defined by Hartwell et al. (43) “cellular functions carried out by “modules” made up of many species of interacting molecules”. The outcome is a summation of all these modules which often interact with each other in a non-vectorial manner.
Integrins are a family of α,β heterodimeric receptors that mediate dynamic linkages between extracellular adhesion molecules and the intracellular actin cytoskeleton. Although integrins are expressed by all multicellular animals, their diversity varies widely among species (49; 73; 94). To date 19 α and 8 β subunit genes encode polypeptides that combine to form 25 different receptors. Integrins have been the subject of extensive research investigating the molecular and cellular basis of integrin function.
Integrins are major contributors to both the maintenance of tissue integrity and the promotion of cellular migration. Integrin-ligand interactions provide physical support for cell cohesion, generation of traction forces in cellular movement, and organise signalling complexes to modulate cellular functions such as differentiation and cell fate. PGs are key ECM components which interact with integrins modifying their function and integrins, in turn, are key regulators of ECM PGs.
Currently little is known about the mechanisms underlying tissue organisation and cellular trafficking, and the regulation of those processes in disease, as well as determining the molecular basis of integrin function. No information has been provided to identify the function of distinct regions within the receptor.
Although numerous reports have employed functional modification approaches using antibodies to β1 integrin, the functional modification by definitions remains obscure since it is mainly focused on activation or blocking of adhesion to a substrate under a defined set of conditions. The limitations of such definition are clear. Firstly, it does not take into account that unlike other receptors, integrins can exist in an inactive, active and active and occupied state. Secondly, the functional modulation is often achieved via different domains and hence may entail different downstream intracellular signalling and therefore even if the effect on adhesion is similar the functional end outcome can be different since each region appears to possess a different function (21; 48; 49; 72). Thirdly, β1 integrin exists in four different splice variants and the difference is in the cytoplasmic domain hence implicating different downstream signalling. The difference in signalling downstream effects between the splice variants is not yet defined. Therefore, the use of functional modification terminology serves best to take the above mentioned points into account since the “blocking” and “activation” of adhesion terminology refers to only one function, of many, of integrin.
Heterodimers of β1 integrin bind collgens (α1,α2), laminins (α1,α2,α3,α7,α9) and fibronectin (α3,α4,α5,α8,αv). It can also act as a cell counter receptor for molecules such as vascular cell adhesion molecule-1 (VCAM-1). Further more, recent reports have demonstrated that b1 integrin can also bind metalloproteinases such as MMP2 (64) and MMP9 (28) and affect their activation state. Both MMPs have been shown to contribute to caspase-mediated brain endothelial cell death after hypoxia-reoxygenation by disrupting cell-matrix interactions and homeostatic integrin signalling (7). TGFβ1 have also been reported to bind to β1 integrin.
The outside-in signaling of integrins is critical to its numerous cellular functions such as adhesion, proliferation, survival, differentiation, and migration. The number and type of integrin receptors heterdimer together with the availability of specific ECM substrates are important in determining which cellular functions are affected. The synthesis and insertion of new integrins into the membrane, removal from the cell surface, or both are possible mechanisms for controlling the number of available integrin receptors. It is possible that new synthesis would require upregulation of expression and sorting of specific a chains to pair with excess β1 in the cytoplasm and presentation of the new α/β heterodimer in a precise location on the cell surface, which is not a very targeted mechanism. An alternative method of regulation could be cleavage at the cell surface, or shedding, as an immediate method for removal of specific integrin-ECM contacts as it would provide a more focused mechanism for regulating specific functions. Furthermore, the shed β1 fragment could bind to cells or ECM components or alternatively be involved in signalling and biological events involved in cellular growth and remodelling. Indeed it has been shown that in myocytes and fibroblasts a change size and shape results in altered cellular contacts with the ECM. This lead to shedding of a β1 integrin fragment from the cell surface (32).
As to the role of β1 integrin in tissue injury and repair, it has been shown to be significantly activated in the infarcted myocardium. Integrin is active particularly at sites of inflammation and fibrosis (90). Integrins- and cytoskeletal-associated cytoplasmic focal adhesion proteins have been suggested to participate in the process of endothelial wound closure where treatment of human coronary artery endothelial cells with anti-β1 integrin function-modifying antibody enhanced wound closure (1). Further in vivo evidence have shown that the loss of β1 integrins in keratinocytes caused a severe defect in wound healing. β1-null keratinocytes showed impaired migration and were more densely packed in the hyperproliferative epithelium resulting in failure in re-epithelialisation. As a consequence, a prolonged inflammatory response, leading to dramatic alterations in the expression of important wound-regulated genes was seen. Ultimately, β1-deficient epidermis did cover the wound bed, but the epithelial architecture was abnormal. These findings demonstrate a crucial role of β1 integrins in wound healing (37).
Apoptosis is a form of cell death that eliminates compromised or superfluous cells. It is controlled by multiple signaling and effector pathways that mediate active responses to external growth, survival, or death factors. Cell cycle checkpoint controls are linked to apoptotic enzyme cascades, and the integrity of these and other links can be genetically compromised in many diseases, such as cancer. The defining characteristic of apoptosis is a complete change in cellular morphology where the cell undergoes shrinkage, chromatin margination, membrane blebbing, nuclear condensation and then segmentation, and division into apoptotic bodies which may be phagocytosed. DNA fragmentation in apoptotic cells is followed by cell death and removal from the tissue, usually within several hours. It is worth noting that a rate of tissue regression as rapid as 25% per day can result from apparent apoptosis in only 2-3% of the cells at any one time.
β1 integrin has also been implicated in apopotosis (76; 77; 101). Involvement of β1 integrin in beta Amyloid Protein (β-AP)-induced apoptosis in human neuroblastoma cells (12). In the presence of either collagen I degrees, fibronectin, or laminin, β-AP toxicity was severely reduced. This protective effect seems to be mediated by integrins, because preincubation of neuroblastoma cells with antibodies directed against β1 and α1 integrin subunits greatly enhanced β-AP-induced apoptosis.
Loss of activity of the β1-integrin receptor in hepatocytes, which controls adhesion to collagen, was seen to precede this loss of adhesive ability. Addition of the β1-integrin antibody (TS2/16) to cells cultured with liver injury serum significantly increased their adhesion to collagen, and prevented significant apoptosis (78). However, this effect seems controversial as experiments with an antibody to integrin β1 suggest that the collagen-chondrocyte interactions are mediated through integrin β1, and these interactions may protect chondrocytes from apoptosis (16).
It has been postulated that prior to the commitment to apoptosis, signals initiated by the apoptotic stimulus may alter cell shape together with the activation states and/or the availability of integrins, which promote matrix-degrading activity around dying cells. This pathway may interrupt ECM-mediated survival signaling, and thus accelerate the the cell death program (64).
Maintenance of the Extracellular Matrix
ECM homeostasis is maintained under normal physiological conditions by a fine balance between degradation and synthesis orchestrated by matrix metalloproteinase (MMPs) and tissue inhibitors of metalloproteinase (TIMPs). This homeostasis is critical in many physiological processes such as embryonic development, bone growth, nerve outgrowth, ovulation, uterine involution, and wound healing. MMPs also have a prominent role in pathological processes such as arthritis (66; 70; 84), chronic obstructive pulmonary disease (17; 92) and atherosclerosis (67). However, little is known about how they are anchored outside the cell.
Mechanical forces are known to modulate a variety of cell functions such as protein synthesis, proliferation, migration or survival and by doing so regulate tissue structure and function. The routes by which mechanical forces influence cell activities have been defined as mechanotransduction and include the tensegrity structure model and signalling through cell surface mechanoreceptors including ECM binding molecules. The tensegrity structure model postulates that a cell maintains a level of prestress generated actively by the actin microfilaments and intermediate filaments (68). This active stress element is balanced by structures resisting compression, mainly microtubules within the cell and components of the ECM. Matrix remodelling in response to mechanical forces is an adaptive response to maintain tensegrity in mechanosensitive tissues including cartilage and lung. In-vivo and in-vitro observations demonstrate that mechanical stimulation is necessary to maintain optimal cartilage and lung structure and function (81; 81; 91; 103). Thus mechanical forces regulate ECM composition which, in turn, will modify the mechanical microenvironment in tissues in a mutually reciprocal manner. This aspect provided a valuable tool for investigating biological functions in vitro.
Extracellular Matrix Catabolism and Anabolism
The ECM provides structural support as well as biological signals to almost every organ in the body. In the lung, the ECM provides structural support and acts as an adhesive as well as a guiding cue for diverse biological processes. Collagens are the most abundant ECM component in the lung constituting 60-70% of lung interstitium followed by elastin and PGs and glycoproteins (96).
The ECM composition of organs varies between the different anatomical and structural sites.
Lung PGs have just recently begun to be characterised. Perlecan and what is thought to be bamacan have been found in all lung basement membranes (20; 74). Of the SLR-PGs, lumican has been shown to be predominant and mainly found in the ECM of vessel walls and to a lesser extent in airway walls and alveolar septa (22). Immunohistochemical studies have demonstrated the presence of biglycan in the peripheral lung, though in very small quantities, where it is associated with airway and blood vessel walls (9; 22; 24). Furthermore, biglycan was shown to be associated with the epithelial cell layer particularly during development. Decorin has been localized to the tracheal cartilage, surrounding blood vessels and airways, and interlobular septae (9). However, Western analyses have demonstrated that decorin expression in the lung parenchyma is undetectable (22). Similarly, it was shown in this study that fibromodulin expression is also undetectable; an observation confirmed by the undetectable mRNA levels for this PG by Westergren-Thorsson et al. (102). The large aggregating PG, aggrecan, is only found in tracheal cartilage associated with HA in a complex stabilized by the link protein (85). On the other hand, versican can be found in small quantities in the airway and blood vessel walls (29), associated with smooth muscle cells (97) and fibroblasts (54), and has been co-localized with elastin fibres (85). HA can be found in tracheal cartilage (85), basolateral surfaces of the bronchiolar epithelium and the adventitia of blood vessels and airways (34; 35). The HA receptor, CD44, is expressed mainly by airway epithelium and alveolar macrophages (57; 62). Syndecans have been reported to be heavily expressed by alveolar epithelial cells (69).
The Importance of the Extracellular Matrix in Disease
Awareness of extracellular matrix importance has been heightened by the recognition that it profoundly influences the biology of the cell and hence, both mechanically and biochemically, the physiology of the whole structure in which the cell is embedded. There may be a real lead to the development of a novel therapeutic intervention where part of the clinical presentation is precipitated by an imbalance in catabolism vs anabolism such as may be found in chronic obstructive pulmonary disease.
Chronic Obstructive Pulmonary Disease (COPD), comprising chronic bronchitis and emphysema, is a major cause of chronic morbidity and mortality throughout the world. In the UK, COPD is the fifth leading cause of death, causing 26,000 deaths and 240,000 hospital admissions annually. The cost to the National Health Service of the UK of COPD-related hospital admissions is in excess of £486 million annually (15). Further costs are incurred due to co-morbidity such as respiratory infections and depression. Research into emphysema pathology and its treatment has been largely neglected because of the view that it is mainly self-inflected. Therefore strategies to effectively manage emphysema are needed in parallel to health promotion.
The Pathology of COPD
COPD is characterised by a progressive and irreversible airflow limitation (95) as a result of small airway disease (obstructive bronchiolitis) and parenchymal destruction (emphysema). Destruction of lung parenchyma is characterised by the loss of alveolar attachments to the small airways, decreased lung elastic recoil and as a consequence diminished ability of the airways to remain open during expiration (8).
Although the main risk factor for COPD is tobacco smoking, other predisposing factors have been identified (86). Emphysema is caused by inflammation, an imbalance of proteinases and antiproteinases in the lung (typified by hereditary α-1 antitrypsin deficiency) and oxidative stress which leads to the destruction of the ECM.
Current Treatments for COPD and Emphysema
To date, the only available drug treatments for COPD sufferers have focussed primarily on bronchodilation using anticholinergics and dual β2-dopamine2 receptor antagonists. Inflammation in COPD is resistant to corticosteroids. Metalloproteinase (MMP) inhibitors are currently being developed to treat COPD, although in their current formulation, serious toxic side effect are almost certain to limit their use. Retinoids have also been shown to induce alveolar repair though this remain largely disputed. However, notwithstanding all such hopeful activities, what is clearly lacking is an agent which may aid in the repair of injured ECM.
In summary, COPD/emphysema is a paradigm for diseases which have a strong element of ECM remodelling as a major contributor to their pathophysiology. Other organs which require tissue repair include, but are not limited to; skin, central nervous system, liver, kidney, cardiovascular system, bone and cartilage. Furthermore, current therapeutics have focused primarily on preventative or symptom-relieving treatments. However, due to the progressive nature of both diseases together with often late diagnosis, regaining normal function remains a problem.
Recently, novel therapeutic approaches targeting integrin function have been adopted. Very late antigen-4 (VLA4) or α4 integrin antagonists are currently in advance stages of trials for the treatment of asthma, multiple sclerosis and Crohn's disease (58; 60; 71). Antagonists to αvβ3 integrin have attenuated adjuvant-induced arthritis and now are undergoing trials (6). The target of the functional blocking or antagonism is attenuating inflammation and this has not been demonstrated to affect the ECM alteration usually associated with those conditions.
The inventors have now surprisingly shown that compounds which modulate the function of beta 1 integrin facilitate improved tissue repair and regeneration.